MINIREVIEW

Beyond conventional antibiotics for the future treatment of methicillin-resistant Staphylococcus aureus infections: two novel alternatives Deirdre Fitzgerald-Hughes1, Marc Devocelle2 & Hilary Humphreys1,3 1

Department of Clinical Microbiology, Education and Research Centre, Royal College of Surgeons in Ireland, Beaumont Hospital, Dublin, Ireland; Department of Pharmaceutical and Medicinal Chemistry, Centre for Synthesis and Chemical Biology, Royal College of Surgeons in Ireland, Dublin, Ireland; and 3Department of Microbiology, Beaumont Hospital, Dublin, Ireland

IMMUNOLOGY & MEDICAL MICROBIOLOGY

2

Correspondence: Deirdre Fitzgerald-Hughes, Department of Clinical Microbiology, RCSI Education and Research Centre, Beaumont Hospital, Smurfit Building, PO Box 9063, Dublin 9, Ireland. Tel.: +353 1 8093711; fax +353 1 809 3709; e-mail: [email protected] Received 8 December 2011; revised 2 March 2012; accepted 2 March 2012. Final version published online 4 April 2012. DOI: 10.1111/j.1574-695X.2012.00954.x Editor: Peter Timms Keywords anti-infectives; antibacterial agents; MRSA; Staphylococcus aureus.

Abstract The majority of antibiotics currently used to treat methicillin-resistant Staphylococus aureus (MRSA) infections target bacterial cell wall synthesis or protein synthesis. Only daptomycin has a novel mode of action. Reliance on limited targets for MRSA chemotherapy, has contributed to antimicrobial resistance. Two alternative approaches to the treatment of S. aureus infection, particularly those caused by MRSA, that have alternative mechanisms of action and that address the challenge of antimicrobial resistance are cationic host defence peptides and agents that target S. aureus virulence. Cationic host defence peptides have multiple mechanisms of action and are less likely than conventional agents to select resistant mutants. They are amenable to modifications that improve their stability, effectiveness and selectivity. Some cationic defence peptides such as bactenecin, mucroporin and imcroporin have potent in vitro bactericidal activity against MRSA. Antipathogenic agents also have potential to limit the pathogenesis of S aureus. These are generally small molecules that inhibit virulence targets in S. aureus without killing the bacterium and therefore have limited capacity to promote resistance development. Potential antipathogenic targets include the sortase enzyme system, the accessory gene regulator (agr) and the carotenoid biosynthetic pathway. Inhibitors of these targets have been identified and these may have potential for further development.

Introduction Serious infections caused by Staphylococcus aureus are important globally in the hospital setting and in the community. These range from minor infections of the skin and soft tissue to life-threatening systemic infections, such as bloodstream infections and endocarditis. Methicillinresistant S. aureus (MRSA) is resistant to most conventional b-lactam antibiotics because of the carriage of the mecA gene encoding an alternative penicillin-binding protein, PBP2a, for which b-lactams have low affinity (Hartman & Tomasz, 1984; Reynolds & Brown, 1985). The majority of MRSA isolates are resistant to drugs in the other antibiotic classes including aminoglycosides and macrolides (Fluit et al., 2001). Our diminishing arsenal of FEMS Immunol Med Microbiol 65 (2012) 399–412

anti-infectives for the treatment of systemic MRSA infections highlights the need for alternative antimicrobial agents with superior properties in terms of efficacy, reduction in toxicity and resistance. Among the agents currently recommended by the Infectious Diseases Society of America for the treatment of MRSA infections are vancomycin, clindamycin, daptomycin, linezolid, trimethoprim, tetracycline and streptogramins (Liu et al., 2011). However, increasingly in vitro resistance to currently used agents is reported and clinical failures have occurred (Soriano et al., 2008; Yoon et al., 2008; Baltz, 2009; Prabhu et al., 2011; Gould et al., 2012; Ruiz de Gopegui et al., 2012). As summarized in Fig. 1, new members of existing antibacterial classes in the late phases of clinical trials, with potential for the treatment ª 2012 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved

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D. Fitzgerald-Hughes et al.

Fig. 1. New MRSA agents in clinical use (*) and MRSA agents in development.

of MRSA infections include ceftobiprole, ceftaroline, dalbavancin, oritavancin (peptidoglycan synthesis inhibitors) and iclaprim (folate synthesis inhibitor). Ceftobiprole and ceftaroline are novel advanced generation cephalosporins with a broad activity spectrum and strong affinity for PBP2a with ceftobiprole showing stability to b-lactamases (Zhanel et al., 2008; Dauner et al., 2010). Dalbavancin and oritovancin are semi-synthetic lipoglycopeptides with a heptapeptide core similar to vancomycin. In addition to effects on the cell wall, these agents also disrupt cell membrane integrity through membrane depolarization. They also have longer half-lives allowing for less frequent dosing compared with vancomycin and teicoplanin (Zhanel et al., 2010). The success of these newer agents remains to be assessed clinically. It is clear that large pharmaceutical companies preferentially appear to favour the development of new generation classical antibiotic classes, with improved properties. This may be because, compared with new agents with alternative mechanisms of action, their safety and efficacy is well established in vivo and they are amenable to pharmaceutical preparation. However, in view of the propensity to develop resistance associated with conventional current antibiotics and their derivatives, the long-term future of anti-staphylococcal agents may involve an exploration of agents with alternative and multiple modes of antibacterial activity. Additional properties such as antipathogenic or immunomodulatory activity would also be desirable in novel MRSA drugs. Such adjunct properties would be particularly important for the treatment of community-associated MRSA (CA-MRSA) which is associated with enhanced virulence that may be toxin mediated (Voyich et al., 2005). The investigation of alternative therapeutic agents with novel mechanisms of action remains largely an activity for academic researchers and small biotechnology companies. This type of research has resulted in preclinical developments in the areas of innate immune defence peptides and antipathogenic agents with potential as novel anti-MRSA therapeutics. For example, cationic peptides offer multiple and alternative modes of action that may circumvent the problem of antimicrobial resisª 2012 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved

tance. Significant improvements, to the chemistry of such peptides, have increased their attractiveness in terms of pharmacokinetics, toxicity and cost. Antipathogenic agents can potentially attenuate the virulence of MRSA, and therefore, this therapeutic approach may have significantly less propensity to contribute to antimicrobial resistance. These novel approaches to the treatment of MRSA infections, though in their infancy in terms of pharmaceutical development, may provide alternative or complementary therapy in the future. Recent developments in these areas and their future potential as novel antiinfectives are discussed.

Cationic host defence antimicrobial peptides and their therapeutic potential Cationic antimicrobial peptides (CAMPs) are a group of ubiquitous peptides that are part of the host innate immune system of animals and plants, and these molecules have several properties that make them promising candidates for development as agents for the treatment of microbial infections including those caused by MRSA (Hancock & Patrzykat, 2002; Zhang & Falla, 2006). Native CAMPs are structurally diverse, varying in size, sequence, content of a helical or b-sheet motifs, disulphide bridges and linear extended structures. Despite their structural diversity, CAMPs are all polycationic and amphipathic, two features thought to facilitate their antimicrobial mechanism (Dathe et al., 1997). The main mechanism of antimicrobial action of host defence peptides (HDPs) is biophysical rather than biochemical, where the target is the cytoplasmic membrane structure itself (Fig. 2). In Gram-positive and Gram-negative organisms, the antimicrobial activity of CAMPs is initiated through electrostatic interactions with the anionic phospholipid head-groups of the cell envelope that may lead to either membrane perturbations as has been shown for human b-defensins (Yeaman et al., 1998) and magainins (Westerhoff et al., 1989) or translocation across the membrane and interaction with various intracellular targets as occurs for cathelicidins such as LL-37 and bactenecin (Sadler et al., 2002). FEMS Immunol Med Microbiol 65 (2012) 399–412

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Fig. 2. Dual effects of host defence peptides. Host defence peptides can exert antibacterial effects directly by forming pores in the cell membrane or can modulate the immune response to infection by inducing transcription of cytokines or directing cellular components of the immune system such as neutrophils, dendritic cells, monocytes and macrophages to the site of infection.

Three host defence peptides have completed or are in phase III clinical trials: the magainin 2 analogue pexiganan (MSI-78) for the prevention of diabetic foot ulcers; iseganan, from pig protegrin, for the treatment of oral mucositis; and omiganan for the prevention of catheter infections and acne. Pexiganan failed to be approved by the FDA because of nonsuperiority to approved agents but it remains one of the best studied CAMPs. Clinical trials involving HDPs have to date mainly been limited to topical applications although some, such as the human lactoferrin fragment hLF1-11, for bacteraemia and fungal infection, being developed for systemic applications are in early clinical trials. The sequences, properties, in vitro activities and phase of development of some of these peptides that may also have potential as S. aureus anti-infectives are outlined in Table 1. Classic antibiotics target biochemical properties such as folate, peptidoglycan, nucleic acid and protein synthesis, which are often mediated through enzyme inhibition or inhibition of binding to intracellular targets. However, the ability of HDPs to kill multi-resistant bacteria and to poorly select resistant mutants may be related to the contribution of additional alternative and multiple pathways to their mechanism of action, such as depolarization of the bacterial membrane, pore formation and the induction of degradative enzymes and disruption of intracellular targets (Hadley & Hancock, 2010). The potential direct antimicrobial activity of mammalian host defence peptides can be complemented by a chemotactic activity for phagocytes and memory and effector T cells (Fig. 1). Additionally, they mediate the FEMS Immunol Med Microbiol 65 (2012) 399–412

recruitment of immature dendritic cells, by direct chemotactic activity or by upregulation of chemokine production in macrophages, and promote maturation of these dendritic cells directly or indirectly by inducing production of inflammatory cytokines (IL-1b, TNFa) (Bowdish et al., 2005, 2006; Yeung et al., 2011). Although these latter activities result in the local release of pro-inflammatory cytokines, host defence peptides can also reduce the systemic production of TNFa, IL-1b and IL-6, as has been demonstrated for LL-37 (Mookherjee et al., 2006). Therefore, HDP modulation of the immune response to bacteria appears to involve not only enhancement of specific pro-inflammatory responses, but also suppression of other elements of the pro-inflammatory response, the additive effects of which contribute to a more controlled inflammatory response after the initial potent cytokine response (Yeung et al., 2011). Some of these immunomodulatory properties alone are sufficient to prevent or clear infection. This was demonstrated by the efficacy in a mouse model of infection, of an immune defence regulator peptide, IDR1, which is devoid of direct antimicrobial activity, but which can selectively activate innate immune responses (Scott et al., 2007). This peptide has recently entered phase I clinical safety trials and is intended for use in the prevention of infection in chemotherapyinduced immune-suppression. More recently, another immune defence regulator, derived from the sequence of the bactenecin peptide IDR-1002, has shown enhanced chemokine induction with a stronger protective effect in an in vivo model of S. aureus infection (Nijnik et al., 2010; Turner-Brannen et al., 2011). The combination of ª 2012 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved

ª 2012 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved

Frog (Rana ornativentris) skin

Frog (Leptodactylus syphax) skin

Derivative of protegrin from porcine neutrophils

Magainan analogue

Defensin peptide mimetic

Derivative of indolicidin from bovine neutophils

Temporin10a

Syphaxin (SPX1-22)

Iseganan (IB-367)

Pexiganan (MSI-78)

PMX-30063D

Omiganan (MBI-266)

VQRWLIVWRIRK

KSRIVPAIPVSLL

ILRWPWWPWRRK

n/a

GIGKFLKKAKKFGKAFVKILKK

RGGLCYCRGRFCVCVGR

GVLDILKGAAKDLAGHVATKVINKI

FLPLASLFSRLL

RIWVIWRR

LLGDFFRKSKEKIGKEFKRIVQRIKDFLRNLVPRTES

TRSSRAGLQWPVGRVHRLLRK

Amino acid sequence

Chemokine induction and enhanced leukocyte recruitment

Chemokine induction and reduction of pro-inflammatory cytokines (Scott et al., 2007)

Membrane depolarization. Inhibition of DNA, RNA and protein synthesis

Membrane disruption

Cell membrane disruption and pore formation

Pore formation, membrane depolarizaton (Sokolov et al., 1999)

Not elucidated

Pore formation, membrane depolarization (Kim et al., 2001)

Membrane depolarisation and cytoplamic permeabilization (Spindler et al., 2011)

Translocation and interaction with intracellular target. Monocyte, T-cell, neutrophil chemotaxis

Translocation and interaction with nucleic acids (Park et al., 1998)

Proposed mechanism

†

*Clinical Laboratory Standards Institute (CLSI) broth microdilution method with modifications, unless indicated otherwise. 90% inhibition, standard CLSI methods. ‡ Units have been converted from µM to mg L 1. § Mean MIC at which 90 % of S. aureus (n = 10) or MRSA (n = 15) isolates were inhibited. ¶ http://irgnews.com/sites/default/themes/publisher/images/companies/PYMX/PYMX-PMX-30063_fs.pdf.

Derivative of bactenecin from bovine neutrophils

Synthetic derivative of bactenecin from bovine neutrophils

Bac8c

IDR-1002

Human (neutrophils and epithelial cells)

LL-37

Derivative of bactenecin from bovine neutrophils

Asian Toad (Bufo bufo gargarizans) stomach

Buforin II

IDR-1

Source

Peptide name

Table 1. Examples of natural cationic peptides with potential for development as Staphylococus aureus anti-infectives

Pre-clinical

31.9‡ (Dourado et al., 2007)

n/a

n/a

16† (Sader et al., 2004)

 2¶

16–64§ (Fuchs et al., 1998)

Pre-clinical

Prevention of infections in the immunecompromised Phase I clinical trials

Prevention of catheter infections and acne. Phase III clinical trials

Acute SSTI. Phase II clinical trials

Topical treatment of diabetic foot ulcers. Phase III clinical trials

Treatment of oral mucositis. Phase III clinical trials

Pre-clinical

0.014‡(Kim et al., 2001)

4 (Mosca et al., 2000)

Pre-clinical

Pre-clinical

Pre-clinical

Stage of development

2 (Hilpert et al., 2005)

31 (Bals et al., 1998)

8 (Giacometti et al., 2000)

†

MIC in mg L 1*

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selective recruitment of effector cells and suppression of inflammatory cytokines found for these peptides would result in a balanced anti-infective response with reduced risk of uncontrolled inflammation.

Cationic host defence peptides with potential as MRSA anti-infective agents Although approximately 17 cationic peptides are in clinical trials to date (though not all in the MRSA therapeutic area) (Yeung et al., 2011), most of these are for topical application. While alternative topical agents may be useful for skin and soft tissue infections, the potential of cationic peptide or HDPs as dual immunomodulatory/bactericidal agents in S. aureus infections may be realized through their development as systemic agents. Two of the best studied natural human HDPs are the cathelicidin, LL-37, and human beta defensin (HBD). These HDPs are released from a variety of cells in response to bacterial challenge. However, it has been suggested that their relatively low in vivo levels and their inactivation by serum constituents are inconsistent with an effective direct killing activity in vivo (Bowdish et al., 2005). As described above, their immunomodulatory activities have been demonstrated and these may be more important than their direct killing properties (Fig. 2). In the area of S. aureus anti-infectives, both LL-37 and HBDs have served as templates for the development of derivatives with improved potential for therapeutic application and lower potential for toxicity than the natural peptides. For example, the combination of HBD with a specific immune-modulatory peptide (mannose-binding lectin) has recently proved effective in a MRSA mouse wound infection model (Li et al., 2010a, b). LL-37 and its synthetic derivatives have shown both in vitro antibacterial activity and inhibition of S. aureus biofilm formation, and no significant haemolysis of erythrocytes (a marker of cell toxicity) was reported up to 100 lg mL 1 of each derivative (Dean et al., 2011). A nonpeptide structural mimetic of defensin, with low toxicity, PMX-30063D, is currently in clinical development for infections involving S. aureus. CAMPs from a wide range of nonhuman sources including pig protegrin, temporins and syphaxins from frog skin and buforin from toad have been investigated for their in vitro activity towards S. aureus including MRSA (Table 1). However, there is further merit in the discovery of peptides of nonhuman and ancient origin because evolutionary dynamics may have driven the modification of effector molecules in early organisms while largely conserving the signalling pathways and pattern recognition systems that respond to infection. Therefore, they have unique structures that may potently activate FEMS Immunol Med Microbiol 65 (2012) 399–412

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specific immune responses that contribute to a more measured inflammatory response, with limited possibility of cross-resistance to natural HDPs. Candidate peptides that may be exploited for specific systemic application for MRSA infections include peptides derived from ancient organisms such as mucroporin and imcroporin from the venom of the scorpion and the recently described c-arminin1a from the eumetazoa Hydra. Mucroporin is a 17 amino acid peptide from the venom of Lychas mucronatus that rapidly kills bacteria by membrane disruption. The native peptide is active against MRSA (MIC = 25 lg mL 1) and other multi-resistant organisms and an improved MIC of 5 lg mL 1 and a broader spectrum of activity have been reported for an amino acid substituted derivative, mucroporin 1 (Dai et al., 2008). Imcroporin is an immune defence peptide from the venom of Isometrus maculates, and in vitro activity has also been demonstrated against MRSA strains (MIC = 20–50 lg mL 1). The peptide demonstrated less than 10 % haemolysis of erythrocytes at the MIC and was comparable to vancomycin in survival studies on mice infected with S. aureus (Zhao et al., 2009). A recombinant 31 amino acid peptide, c-arminin 1a, from the ancient fresh water animal of the Eumetazoa species, Hydra magnipapillata, has been recently shown to have potent anti-MRSA activity in vitro (0.4 lM), does not demonstrate haemolytic activity and its activity is independent of the salt concentration (Augustin et al., 2009). In sequence, this peptide does not resemble any known protein and it lacks cysteine residues, which would facilitate its synthesis and production in large quantities. These properties make c-arminin an attractive molecule for further exploitation. The search for ancient cationic peptide structures with potent activity towards multi-resistant clinically important bacteria such as MRSA is on-going but has already revealed potential candidates that may serve as lead compounds.

Challenges in developing host defence peptides as therapeutic agents The major obstacles to the development of cationic peptides as systemic therapeutics are concerns about their potential toxicity or immunogenicity and their poor stability. In addition, concerns about development of peptide resistance and unknown effects of synthetic HDPs on the natural innate response to infection have been raised. Host defence peptides are expensive to produce in commercial quantities, and this issue has also affected their potential for development. The relative lack of negatively charged lipids on mammalian cell surfaces and their weak membrane potential gradient may selectively protect eukaryotes from the action of cationic peptides. However, some cationic ª 2012 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved

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peptides such as LL-37 can translocate across mammalian cell membranes because their sequence resembles that of nuclear signalling peptides. Limited data are available on the cytotoxic effects of cationic peptides on mammalian cells. LL-37 does not show significant haemolytic activity at concentrations greater than its antimicrobial activity but in vitro cytotoxic effects have been reported that are dependent on the nature and metabolic state of the target cells and on the evolutionary form of the mature peptide. (Tomasinsig et al., 2009). Because of their small size and linear structure, the majority of host defence peptides are considered to be weakly immunogenic but antibodies have been successfully raised against some cationic peptides such as defensins, hCAP-18 and lactoferrin (Panyutich et al., 1991; Shimazaki et al., 1996; Sorensen et al., 1997). In vivo toxicity is an area that has not been systematically assessed for cationic host defence peptides, and this may be because so few have proceeded to this level in clinical trials. It has been suggested that HDPs, if developed as MRSA anti-infectives, would have low propensity to select resistant mutants compared with classical antibiotics. This is based on the multiple mechanisms of action of HDPs. However, bacteria and the human host have co-evolved, and S. aureus adaptations have been described for a small number of host defence peptides. For example, reduced susceptibility to defensin and protegrins has been demonstrated in S. aureus which is mediated by the incorporation of positively charged L-lysine into the cytoplasmic membrane and is catalysed by the product of the mprF gene (Peschel et al., 2001; Ernst et al., 2009). Interestingly, this membrane modification also contributes to S. aureus resistance to the CAMP-like agent daptomycin, which is currently in clinical use for MRSA infections. An investigation of the evolution of CA-MRSA shows that USA 300 and USA500 strains are more resistant to the innate immune defence peptides, dermicidin and indolicidin than isolates from the epidemic clones from which they originated (Li et al., 2009). Despite these reported resistances, the immune-modulatory properties of HDPs, which may arguably be more important than their direct antimicrobial therapeutic properties, are not influenced by conventional resistance mechanisms and this is where HDPs may offer a real advantage over conventional antibiotics. Natural HDPs may be released either locally at the site of infection or systemically in response to infection (Yang & Oppenheim, 2004). Some authors have argued that the augmentation of these triggers or the provision of analogous triggers of host immunity may dampen the natural innate or adaptive responses to infection or may cause excessive stimulation of inappropriate immune responses. Inappropriate antibody responses to the administration of ª 2012 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved

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self-proteins have been infrequently reported. The possibility of unpredictable effects on the natural host immune response highlights the importance of detailed characterization of the innate immune response. These investigations would include characterization of signalling pathways of pattern recognition agonists, regulatory elements of innate immunity and selective immunomodulatory effects of HDPs. Development of host defence peptide-based agents for systemic administration will require considerable efforts to overcome some of the limitations mentioned above. However, improvements that address some of the limitations of promising candidate peptides have been reported. Substitution of D-amino acids into the peptide sequence of LL-37 derivatives was shown to minimize proteolysis and increase antibacterial activity (Stromstedt et al., 2009), and the in vitro cytotoxic effects of LL-37 have been reduced by truncation of the sequence while antibacterial activity is retained (Nell et al., 2006). Modifications that increase overall charge or amphipathicity increase potency, allowing lower concentrations to be used (Chen et al., 2005). Pharmacokinetic properties have improved with the conversion of some host defence peptides to peptidomimetic or peptoid forms, use of D- or b- amino acids and PEGylation (Hong et al., 1999; Hamamoto et al., 2002; Hancock & Sahl, 2006; Imura et al., 2007). Some of these host defence mimics, in addition to their excellent drug-like properties, failed to generate resistant derivatives of S. aureus in vitro compared with ciprofloxacin or norfloxacin (Tew et al., 2006). Targeted delivery of host defence peptides to the site of infection may further improve the therapeutic potential of these molecules. The increased local concentrations that could be reached with this approach could potentially remove constraints because of higher relative MICs for some HDPs. Improved delivery has had some success in the area of host defence peptides as candidates for anticancer therapy, including conjugation to a ‘tumourhoming’ motif, peptide hormone or antibody, bioconversion to an active agent by tumour-specific enzymes and liposomal technology (Ellerby et al., 1999; Marks et al., 2005; Mader & Hoskin, 2006; Chakrapani et al., 2008; Jia et al., 2008; Song et al., 2009a). The identification and assessment of similar targeting approaches for delivery of defence peptides to sites of infection is in its infancy with antibody conjugation of a synthetic derivative of a salivary host defence peptide, histatin serving as an example. While pro-peptide inactivity in this case has not been clearly demonstrated, with improved design, the approach has clear therapeutic potential (Szynol et al., 2006). In the MRSA field, the further development of cationic peptides for systemic use as targeted candidates against MRSA will depend on the selection of appropriate FEMS Immunol Med Microbiol 65 (2012) 399–412

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effective candidates that are amenable to chemical modification and the design of bacterial or site of infectionmediated targeting approaches. Another limitation to the therapeutic application of peptide-based anti-infectives is the high cost associated with chemical synthesis in large quantities. Synthetic mimics of antimicrobial peptides that have an unnatural backbone but maintain the biophysical characteristics of CAMPs offer a cost advantage (Rotem & Mor, 2009). Recently, the economic feasibility of chemical synthesis on a multi-tonne scale has been demonstrated for the biomimetic antiretroviral agent, enfuvirtide (Bray, 2003). Large-scale recombinant production of the fungal defensin, plectasin, has been achieved at commercially viable yield and purity, from cultures of the yeast, Aspergillus oryzae (Mygind et al., 2005). Methodologies for largescale industrial production of seven recombinant host defence peptides representative of those that are currently undergoing clinical trials have recently been developed by fusion to sumoase protease (SUMO), cloning into Escherichia coli and a two-step purification of the fusion product from the culture. This expression system gave high yields of intact and biologically active peptides and has demonstrated a cost-effective means of HDP production under good laboratory manufacturing processes that would be required for human therapeutic applications (Bommarius et al., 2010).

Therapeutic approaches that target MRSA virulence Another novel approach to the development of antistaphylococcal agents with reduced capacity to elicit bacterial resistance is the development of ‘antipathogenic’ agents. These agents are designed to interfere with bacterial virulence mechanisms including binding to host tissues, evasion of phagocytosis, biofilm production and the production of toxins. The limited antibacterial activity of such agents may minimize the development of resistance while controlling the pathogenic process through diminished bacterial virulence. Controlling pathogenic processes in this way may allow the host immune response to more effectively overcome the infection. However, these agents could serve as adjuncts for immunocompromised patients. This antipathogenic approach, which relies on the identification and characterization of appropriate virulence targets, has been an academic research pursuit for over two decades. Promising targets that may be disrupted among S. aureus in the development of novel antipathogenic drugs include the accessory gene regulator (agr), sortase enzyme system, the carotenoid biosynthetic pathway and other recently discovered regulatory pathways. These systems contribute to the ability of S. aureus FEMS Immunol Med Microbiol 65 (2012) 399–412

to effectively invade and damage the host, and therefore, their modulation represents a novel strategy in the antiinfective field and should be further explored.

The quorum sensing response The quorum sensing response in S. aureus describes the coordinated expression of virulence genes in response to bacterial cell density and is modulated by complex regulatory systems, the best characterized of which is the accessory gene regulator (agr). Agr modulation contributes to the expression of a variety of virulence genes at different stages of infection through quorum sensing auto-inducing peptide (AIP) signals. (Novick, 2003; Cheung et al., 2004). This role for agr in the inverse coordinated expression of genes that promote colonization and invasion has prompted many researchers to pursue agr as an antivirulence target. Specific molecules in the agr system, AIP and RNAIII (the effector molecule), have been investigated as potential targets for inhibition (Dell’Acqua et al., 2004; Qazi et al., 2006; Balaban et al., 2007; George et al., 2008). A global inhibitor of S. aureus AIPs was designed based on structure–function analysis and consists of a truncated thiolactone region of AIP-II (Lyon et al., 2000), and more recently, investigations of a series of synthetic mimetics of this region have revealed the minimum structural requirements for inhibition (George et al., 2008). Early administration of an AIP analogue attenuated abscess formation in a mouse subcutaneous abscess model but based on their findings, the authors suggest that administration of such quorum sensing inhibitors for S. aureus infections may be only of prophylactic value based on the kinetics of AIP activation (Wright et al., 2005). The potential of targeting agr for the treatment of device-related infections, which are difficult to treat with conventional antibiotics because of biofilm production, has been demonstrated by inhibition of this regulatory system with RNAIII-inhibiting peptide (RIP). This peptide caused a significant dose- and duration-dependent reduction in bacterial load in MRSA graft infections in rats, which was further reduced when RIP was administered in combination with teicoplanin (Balaban et al., 2007; Simonetti et al., 2008). The therapeutic efficacy and safety of RIP and two synthetic analogues of RIP have also been shown in histopathological studies in a mouse model of S. aureus sepsis (Ribeiro et al., 2003). Although the target of RNAIII activating peptide has controversially been shown not to function in S. aureus pathogenesis (Shaw et al., 2007), RIP has been shown to reduce staphylococcal infection in several in vivo models of infection and no toxicity has been noted. With regard to biofilm dispersal, however, it has conversely been shown in vitro ª 2012 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved

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that agr inhibition is required for biofilm formation, and biofilm dispersal has been demonstrated with the addition of AIP to up-regulate agr-induced protease production (Boles & Horswill, 2008). More recently, the nonribosomal secondary metabolite, aureusamine, was reported to regulate virulence gene expression, and the isogenic ausA mutant, which failed to haemolyse blood agar, had attenuated virulence in a mouse model of infection compared with the wild-type strain (Wyatt et al., 2010). This reported role for aureusamine in virulence gene regulation was later found to be because of an inadvertent mutation in the SaeR two component regulator system (Sun et al., 2011). The controversies surrounding the genetic stability of the agr locus in laboratory strains and the complexity of the roles of RIP, AIP and aureusamine in S. aureus pathogenesis have hampered progress in targeting quorum sensing systems for the discovery of novel anti-infectives. Nonetheless, these studies have been important in demonstrating the therapeutic potential of targeting virulence mechanisms and have prompted the study of other pleiotrophic regulators. With regard to novel therapeutic agents to inhibit agr-mediated virulence expression, the discovery of new molecules may be advanced due to the development of a simple, inexpensive assay to allow screening of large numbers of molecules for their effects on S. aureus virulence. This system is based on the observation of colour changes in response to the candidate molecule, in the growth media of S. aureus strains with lacZ fusions to the agr-regulated genes, spa and hla, in the presence of a beta-galactosidase substrate (Nielsen et al., 2010).

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Staphylococcus aureus sortase enzymes Attachment of S. aureus to host endothelial tissue is facilitated by proteins that recognize specific tissue components such as fibrinogen, fibrin and collagen. The activity of these so-called microbial surface components recognizing adhesive matrix molecules (MSCRAMMS) is dependent on their covalent attachment to bacterial peptidoglycan. The anchoring of these molecules to the cell wall is catalysed by a group of cysteine transpeptidases called the sortase enzymes (Fig. 3), which in S. aureus include two isoforms, SrtA and SrtB (Mazmanian et al., 1999, 2002). SrtA is constitutively expressed, while SrtB is expressed in response to low iron conditions. Deletion of the sortase A gene (srtA) in S. aureus results in failure to display MSCRAMMs and therefore attachment to host components including IgG, fibronectin and fibrinogen. In a mouse model of S. aureus infection, mutants lacking srtA had at least a 2 log reduction in bacterial growth in multiple organs and a 1.5 log increase in lethal dose compared with the wild type (Mazmanian et al., 2000). Later investigations demonstrated that srtA knockout mutants showed reduced virulence in models of septic arthritis and endocarditis (Jonsson et al., 2003; Weiss et al., 2004). It has been recently shown that disruption of srtA in five biofilm-producing clinical isolates of MRSA results in significant reduction (up to sixfold) in glucose-induced biofilm formation which can be reversed by complementation (O’Neill et al., 2008). The SrtB enzyme has a role specifically in the attachment of iron acquisition proteins such as IsdA, isdB, etc, and mutants that lack the SrtB gene are also associated with reduced virulence in the

Fig. 3. Mechanism of sortase processing of MSCRAAMs. Sortase B cleaves at the LPXTG motif to allow display of MSCRAAMs at the cell surface. Their display facilitates adhesion to host cells. If sortase is inhibited, the bacterial cell has reduced adhesion to the host cell as the surface adhesins are not displayed.

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mouse model of septic arthritis but only in the later stages of infection when iron is limited in the environment (Jonsson et al., 2003). The pathogenesis of S. aureus in persistent infections is linked to its ability to survive within macrophages where it is protected from the host immune response. Expression of SrtA has also been shown to be critical to phagosomal survival of S. aureus as SrtA mutants are efficiently killed by macrophages (Kubica et al., 2008). These studies suggest that SrtA specifically may be a potential target for the development of novel anti-infective agents and may have specific application for complicated or persistent S. aureus infection including those involving biofilms. Selective toxicity by sortase inhibition is possible as there is no related sortase homologue in eukaryotic cells. The localization of SrtA within the cell membrane of S. aureus and other Gram-positive organisms offers an advantage in terms of the ease of access to this target where the activity of potential inhibitors will not rely on transport across the cell envelope. It has been speculated that bacterial resistance to sortase inhibition would be reduced compared with classical antibiotics given that SrtA mutants have similar growth rates to the wild type (Weiss et al., 2004). The lack of disruption to essential gene function by SrtA mutation or inhibition, together with significantly attenuated virulence potential associated with loss of sortase activity, suggests that selective pressure would not be as significant for these possible agents as it is for antibiotics such as penicillin or aminoglycosides where the target is essential for cell survival and where selective pressure would favour the development of resistance. Numerous molecules have been investigated as potential inhibitors of sortase enzymes. Some of the most promising of these have been discovered by small molecule screening and were selected based on their ‘drug-like’ structures. For example, Oh and colleagues discovered a novel class of S. aureus sortase inhibitors, the diarylacrylonitriles, from a library of 1000 small molecules. Modification of the lead compound from the initial screen resulted in a reduction in IC50 from 231 to 9.244 lM (Oh et al., 2004). These authors have further shown that this molecule, (Z)-3-(2,5-dimethoxyphenyl)-2-(4-methoxyphenyl) acrylonitrile, was effective in an in vivo mouse model of S. aureus infection. Survival rates increased and joint and bone infections decreased in the treated animals compared with controls (Oh et al., 2010). The aryl (b-amino) ethyl ketones were also selected from a large screening library of small molecules. These are mechanism-based enzyme inhibitors that have selectivity for S. aureus SrtA with IC50 and Ki values in the low micromolar range (lead compounds IC50 15–47 lM) (Maresso et al., 2007). More recently, pyridazinone and pyrazolethione analogues, selected from over 300 000 small molFEMS Immunol Med Microbiol 65 (2012) 399–412

ecules, have been shown to reversibly inhibit SrtA with IC50s in the high nanomolar range (Suree et al., 2009). Sortase remains an attractive candidate as an antivirulence target, and the discovery of several distinct sortase inhibitors with activities in the nano- to micro-molar range and with drug-like properties is encouraging. However, challenges remain that require further investigation. The inhibition of sortase enzymes, by preventing the display of surface antigen, may dampen the host immune response which is required for bacterial clearance. Furthermore, bacterial clearance, even for virulence attenuated bacteria, requires active opsonophagocytic killing which may be impaired in the immunocompromised patient. A pharmacological evaluation of sortase inhibitors should be carried out, to assess therapeutic efficacy and toxicity. Further discoveries are needed to increase the pool of molecules available for further investigation as potential therapeutic agents. The further advancement of these discoveries will be initially guided by their properties in in vivo models of infection.

Staphyloxanthin biosynthesis The antioxidant properties of the carotenoid pigment, staphyloxanthin, responsible for the golden colour of S. aureus, protect the organism from reactive oxygen species produced by neutrophils (Liu et al., 2005). This finding suggests that modulation of this metabolic pathway may have antipathogenic effects. In a mouse subcutaneous model of infection, mice infected with S. aureus mutants lacking this pigment have significantly reduced bacterial loads and no visible lesions compared with the wild-type strain (Liu et al., 2005). Increased bacterial clearance of staphyloxanthin mutant compared with the wild type was also shown by these authors in a murine model of nasal colonization (Liu et al., 2008). One of the key enzymes in staphyloxanthin biosynthesis is S. aureus dehydrosqualene synthase (SQS or CrtM) which catalyses the condensation of two molecules of isoprenoid farnesyl disphosphate to form dehydrosqualene. Interestingly, there is overlap between the early steps of staphyloxanthin biosynthesis and human cholesterol biosynthesis. Human SQS and the bacterial enzyme CrtM have 30 % sequence identity but have been shown to share significant structural features (Liu et al., 2008). Furthermore, compounds originally developed as cholesterollowering agents have been shown to inhibit S. aureus CrtM in the nanomolar range and have been investigated as potential antipathogenic agents (Liu et al., 2008). Two cholesterol-lowering agents, lapaquistat acetate and squalestatin, interact with both human squalene synthase and S. aureus CrtM at specific common residues (Kahlon et al., 2010). Among the most potent inhibitors of CrtM ª 2012 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved

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that also prevent staphyloxanthin formation in cellular assays are the phosphosulphonates with Ki in the range 1.5–135 nM and the diphenyl ether phosphonoacetamides with Ki in the range 30–70 nM. The most potent of the phosphosulphonates (designated BPH652) was further tested because it had advanced through preclinical animal testing and two human clinical trials as a cholesterollowering agent (Sharma et al., 1998a, b). No inhibition of the growth of three human cell lines was found up to a concentration of 300 lM BPH652. The in vivo activity of BPH652 has also been determined in a mouse model of systemic S. aureus infection, and 98% reduction in S. aureus colony forming units was achieved in the treated group (Liu et al., 2008). The question of selectivity for the S. aureus CrtM over human SQS has also been addressed by these authors and several halogen-substituted derivatives show selectivity for the bacterial enzyme (Song et al., 2009b). The diphenyl ether phosphonoacetamides have further improved properties in terms of their uptake into cells (IC50 = 8 nM) while retaining their selectivity for the bacterial enzyme and their negligible toxicity in human cell lines (Song et al., 2009c). The inhibition of the staphyloxanthin pathway in S. aureus, as antivirulence agents, is attractive, because many cholesterol-lowering agents have previously undergone clinical trials, and their toxicities and pharmacokinetic properties are already known (Liu et al., 2008). Further testing of the improved molecules described above in animal infection models will be eagerly awaited. The pigmentation of S. aureus because of staphyloxanthin can be exploited in the development of technologies for rapid screening of candidate inhibitory molecules and one such system has been used successfully to identify at least four known inhibitors of lipid metabolism that reduce staphyloxanthin pigmentation, from a natural compounds library (Sakai et al., 2012).

Conclusion The anti-infectives industry appears to rely on the development of further generations of conventional antibiotics which have improved properties but do not offer new modes of action. Here, we have highlighted areas where basic and applied research has demonstrated the potential of novel anti-MRSA therapies. It is clear, however, that further research is required to determine when and how these compounds can be administered. Investment in generating convincing in vivo data that support a protective role for novel therapeutic agents with minimum sideeffects is required. Given that the majority of patients requiring therapeutic intervention for S. aureus infection are immunocompromised, it appears that both of the ª 2012 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved

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approaches discussed here would have potential as adjuvant therapies rather than their exclusive use as antiinfectives. It is interesting therefore that synergistic in vitro and in vivo effects have been reported using a combination of two HDPs and vancomycin (Cirioni et al., 2006), and a potential advantage of the administration of pexiganan with b-lactam antibiotics has also been demonstrated (Giacometti et al., 2005). These combined applications would potentially extend the therapeutic effectiveness of current antibiotics. Antivirulence approaches, aimed at modulating the pathogenic effects of S. aureus infection, could also be investigated in conjunction with conventional antibiotics.

Transparency declaration HH has had recent research collaborations with Steris Corporation, 3M, Inov8 Science, Pfizer & Cepheid. He has also recently received lecture & other fees from 3M, Novartis & Astellas. DF, MD none to declare.

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